Process for the production and purification of the collagenase enzyme from Vibrio alginolyticus

ABSTRACT

The present invention claims a novel process for the production and purification of microbial collagenase (Microbial Collagenase EC 3.4.24.3) produced by the non-pathogenic aerobic bacterium Vibrio alginolyticus chemovar. iophagus (NCIMB Number: 11038, synonym LMG 3418, hereinafter called Vibrio alginolyticus), which said process provides high production levels of collagenase with a stable, reproducible, cheap fermentation process. The collagenase produced from Vibrio alginolyticus according to the process described herein also presents a specific activity superior to that of other microbial collagenases, is stable in aqueous solution, and can be frozen without significant damage. A further subject of the present invention is pharmaceutical compositions containing collagenase obtained according to the production and purification process described, for the purpose of therapeutic treatment of disorders characterised by collagen accumulation or for the treatment of blemishes/imperfections that benefit from reducing local collagen accumulations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 15/647,999,filed on Jul. 12, 2017 (and issued as U.S. Pat. No. 10,774,319), whichis a Divisional of copending application Ser. No. 14/394,481, filed onOct. 15, 2014 (and issued as U.S. Pat. No. 9,738,883), which was filedas PCT International Application No. PCT/EP2013/057998 on Apr. 17, 2013,which claims priority under 35U.S.C. § 119(a) to Patent Application No.PD2012A000118, filed in Italy on Apr. 18, 2012, all of which are herebyexpressly incorporated by reference into the present application.

FIELD OF INVENTION

Collagenases are metalloenzymes with a proteolytic activity whichrequire the zinc ion in the active site in order to perform theirspecific function of breaking down native collagen. Unlike otherproteases, they can hydrolyse collagen under physiological pH andtemperature conditions. A number of collagenases produced by bacteriaare known in the prior art (Vibrio, Clostridium, Streptomyces,Pseudomonas); those produced by Clostridium (Santyl®, Noruxol®) arewidely used in pharmaceutical compositions for the treatment of skinulcers of various origins, bedsores, burns of different degrees andhypertrophic scars, because they break down the collagen present in thenecrotic tissue. This facilitates the removal of cell debris, whichoften constitutes an obstacle to the migration of epithelial cellsduring the wound-healing and re-epithelialisation process (Rao, D. B. etal., 1975). Collagenase from Vibrio was also used recently for similarpurposes (EP1901755); this patent describes solely lipophiliccompositions (lipogels) containing carrageenan as stabilising agent.However, the inflammatory role generally played by that substance isknown to the skilled person. In any event, regardless of its origin,collagenase has extremely low stability in an aqueous carrier, and istherefore always formulated in wholly lipophilic carriers; Santyl®,Noruxol® and Bionect Start® are ointments with a wholly lipophilic basein which the enzyme is never distributed evenly, and is subject to asignificant loss of activity over time.

The lipophilic carrier ensures the stability of the enzyme and thestorability of the pharmaceutical product, while penalising itstherapeutic activity; collagenase is released from the lipophiliccarrier very slowly, with the result that its bioavailability is greatlylimited. Collagenase can also be used for the systemic treatment, inparticular by injection, of disorders such as adhesive capsulitis(frozen shoulder), Dupuytren's contracture, Peyronie's disease,cellulitis and post-surgical adhesions. Stability in the aqueous carrieris crucial for these applications. A product for injective treatment ofDupuytren's contracture is currently on the market (a lyophilicsubstance reconstituted at the time of use) which contains a mixture oftwo collagenases in a precise weight ratio, which are extracted andpurified by fermentation of the bacterium Clostridium histolyticum.

As already stated, the latter is one of the most common sources ofcollagenase; however, it is a pathogenic micro-organism, which needsanaerobic fermentation (Mandl, I. et al., 1958, Arch Biochem Biophys,74:465-475).

The collagenolytic activity of some strains of Achromobacter wasidentified for the first time in 1972 (Thomson, J. A., Woods, D. R. andWelton, R. L. 1972), and the first studies were subsequently conductedon the strain Achromobacter iophagus (subsequently reclassified asVibrio alginolyticus chemovar. iophagus, Emod, I. et al. 1983) whichdemonstrated the presence of a collagenase with high specific activity(Welton and Woods 1973). As regards the choice of micro-organism to beused to produce collagenase by fermentation, the use of Vibrioalginolyticus is much more advantageous than Clostridium histolyticum,because it is non-pathogenic and allows the fermentation to be performedin an aerobic environment, with considerable industrial advantages. Thepathogenicity of the microbial strain is an important aspect, becauseany impurities (microbial or protein residues) in the finished productcan give rise to serious side effects. Working with pathogenic strainsobviously requires particular care at the purification stages, andconsequently a more complex, expensive industrial process.

The microbial collagenase EC 3.4.24.3 produced from Vibrio alginolyticusis a Zn²⁺ metalloprotease which is also distinguished from thecollagenases produced by Clostridium histolyticum for a number ofreasons:

-   -   it acts specifically on the synthetic peptide        Pz-Pro-Leu-Gly-Ala-D-Arg (where PZ=4-phenyl azobenzyl        oxycarbonyl), which is the synthetic substrate of choice for the        evaluation of collagenolytic activity. The collagenase from V.        alginolyticus produced according to the present invention is the        only one able to break down collagen at the Leu-Gly bond (Keil,        B., Gilles A.-M., Lecroisey, A., Hurion, N. and Tong, N.-T.        1975; Keil B. Matrix Suppl. 1992; 1:127-33);    -   it has much greater proteolytic activity than the analogue        obtained from Clostridium histolyticum;    -   it has a specificity at the cleavage site on native collagen; it        cleaves the helical chain of native collagen at 2 sites,        preferably at ¾ from the N-terminal end at the Y-Gly bond of the        Pro-Y-Gly-Pro sequence, where Y is a neutral amino acid.        Conversely, collagenase from Clostridium histolyticum presents        various cleavage sites in the native collagen chain        (Lecroisey, A. Keil, B. 1979);

The Vibrio alginolyticus chemovar. Iophagus strain only produces onecollagenase, although SDS-PAGE analysis of the products of purificationdemonstrates the presence of several bands with different molecularweights where collagenolytic activity is maintained. These low molecularweight species have been attributed to a process of autoproteolysis ofthe enzyme which is inhibited by specifically formulated buffers(Keil-Dlouha, V. 1976).

In 1992, Takeuchi et al. cloned the entire sequence encoding forcollagenase from Vibrio alginolyticus. The amino-acid sequence deducedfrom the nucleotide sequence shows that mature collagenase is formed by739 amino acids with a molecular weight of 81,875 Da. The nucleotide andamino-acid sequences of collagenase from Vibrio alginolyticus do notexhibit any significant similarities with those of other collagenases(Takeuchi, H., 1992). To date, processes for the production of theenzyme from the strain in question have produced rather modest yieldsand products with an unsatisfactory degree of purity, especially forinjectable use. Patent EP 0115974 describes a process for the productionand purification of collagenase from V. alginolyticus; the final productis a mixture of enzymes (collagenase, neutral proteases andendonuclease) which is only stable after the addition of bovine skincollagen fragments (ASF).

The material obtained has a very low degree of purity. Moreover, thepresence of ASF can create problems at the time of preparation of thepharmaceutical forms identified or, in particular, as it is a materialof animal origin, when the chosen pharmaceutical compositions areadministered by injection.

Moreover, as already stated, the pharmaceutical compositions currentlyknown take the form of an ointment, whereas for topical applicationsand, above all, for systemic applications, it is essential for thecollagenase enzyme to be in extremely pure form and stable in an aqueouscarrier (to improve the distribution of the enzyme in the composition);an aqueous carrier is absolutely preferred for injection treatment.

The present invention overcomes these problems by disclosing aninnovative process for the production and purification of the enzymecollagenase from V. alginolyticus, a process characterised by highyields, reproducibility, stability and a high degree of purity of thefinished product. The finished product is also stable in aqueoussolution and can therefore be stored for long periods, even attemperatures ranging between −20 and −80° C., without undergoingsignificant damage.

Due to the high degree of purity and the specificity of cleavage on thecollagen chain, the collagenase claimed herein can also be used todissociate tissues and isolate cell clusters or single cells for allexperimental and therapeutic procedures requiring isolated cells.

FIG. 1 is a global spectrum of a tryptic digest analysis.

FIG. 2 is a flow chart of the production and purification process of thepresent invention.

FIG. 3 is a SDS-PAGE of the 300K and 10K ultrafiltration stages of thepresent invention.

FIG. 4 is a typical chromatogram of a DE-52 resin.

FIG. 5 is a SDS-PAGE of a peak from a typical chromatogram of a DE-52resin containing collagenase.

FIG. 6 is a typical chromatogram of Source™15Q.

FIG. 7 is a SDS-PAGE of a fraction of Source™15 Q.

FIG. 8 is a SDS-PAGE of the final product of the present invention.

FIG. 9 is a photograph of a 1.5 ml Corning Costar SpinX tube (A), acontainer bearing a membrane with a porosity of 0.22 μm (B), and a tubewith a substrate solution (C).

FIG. 10 illustrates the evaluation of collagenolytic activity in thepresent invention.

FIG. 11 provides four graphs showing the pre- and post-sterilizationrheological evaluation of the present invention.

FIG. 12 provides a graph showing the collagenase activity of differentformulations over time for daily application.

FIG. 13 provides a graph showing the collagenase activity of differentformulations over time for application every two days.

FIG. 14 provides a graph showing the collagenase activity of differentformulations over time for daily application.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims a novel process for the production andpurification of microbial collagenase (Microbial Collagenase EC3.4.24.3) produced by the non-pathogenic aerobic bacterium Vibrioalginolyticus chemovar. iophagus (NCIMB Number: 11038, synonym LMG 3418,hereinafter called Vibrio alginolyticus); said process provides highproduction levels of collagenase with a stable, reproducible, cheapfermentation process. The sequence of collagenase produced from Vibrioalginolyticus according to the production and purification processherein described (SEQ ID NO: 1) is characterized by the deletion of theamino acids 1-75 in comparison with the sequence of 814 amino acidsencoded by the gene of the collagenase from V. alginolyticus (hereinreported as SEQ ID NO: 2 corresponding to Microbial collagenase EC3.4.24.3). By the process of the invention, the mature protein, that isthe active core, consisting of 739 aa, more precisely the 76-814 aa, isproduced:

(SEQ ID NO: 1) TACDLEALVTESSNQLISEILSQGATCVNQLFSAESRIQESVFSSDHMYNIAKHTTTLAKGYTGGGSDELETLFLYLRAGYYAEFYNDNISFIEWVTPAVKESVDAFVNTASFYENSDRHGKVLSEVIITMDSAGLQHAYLPQVTQWLTRWNDQYAQHWYMRNAVNGVFTILFGGQWNEQFVQIIGNQTDLAKALGDFALRASSIGAEDEFMAANAGRELGRLTKYTGNASSVVKSQLSRIFEQYEMYGRGDAVWLAAADTASYYADCSEFGICNFETELKGLVLSQTYTCSPTIRILSQNMTQEQHAAACSKMGYEEGYFHQSLETGEQPVKDDHNTQLQVNIFDSSTDYGKYAGPIFDISTDNGGMYLEGDPSQPGNIPNFIAYEASYANADHFVWNLEHEYVHYLDGRFDLYGGFSHPTEKIVWWSEGIAEYVAQENDNQAALETILDGSTYTLSEIFETTYDGFDVDRIYRWGYLAVRFMFENHKDDVNQMLVETRQGNWINYKATITQWANLYQSEFEQWQQTLVSNGAPNAVITANSKGKVGESITFSSENSTDPNGKIVSVLWDFGDGSTSTQTKPTHQYGSEGEYSVSLSVTDSEGLTATATHTVVISALGGNDTLPQDCAVQSKVSGGRLTAGEPVCLANQQTIWLSVPAVNESSNLAITTGNGTGNLKLEYSNSGWPDDTNLHGWSDNIGNGECITLSNQSNYWGYVKVSGDFENAAIVVDFDAQKCRQ

Furthermore the collagenase produced from Vibrio alginolyticus accordingto the process of the invention also presents a specific activitysuperior to that of other microbial collagenases, is purer and stable inaqueous solution, and can be frozen without significant damage.

Therefore a further object of the present invention is the collagenase,obtained according the described production and purification process,for the use in the therapeutic treatment of pathologies characterized bythe accumulation of collagen or in the treatment ofblemishes/imperfections that benefit from reducing local collagenaccumulations; such as for example, it is worth to mention, skin ulcersof various origins, bedsores, burns of different degrees, scalds, andhypertrophic scars, cellulitis, post-surgical adhesions and still“frozen shoulder” or adhesive capsulitis, Dupuytren's contracture,Peyronie's disease.

A further object of the present invention is pharmaceutical compositionscontaining collagenase obtained according to the described productionand purification process, for the use in the therapeutic treatment ofdisorders characterised by collagen accumulation or for the treatment ofblemishes/imperfections that benefit from reducing local collagenaccumulations; examples are skin ulcers of various origins, bedsores,burns of different degrees, scalds, and hypertrophic scars, cellulitis,post-surgical adhesions, adhesive capsulitis (frozen shoulder),Dupuytren's contracture and Peyronie's disease.

The collagenase obtained as described herein is also suitable forapplication in tissue dissociation and isolation of cell clusters orsingle cells. This application is used, for example, in the Langerhansislet cell transplantation procedure to isolate the islet cells from thesurrounding pancreatic tissue, and in general in all experimental andtherapeutic procedures requiring tissue dissociation.

The collagenase obtained by the process described below is characterisedby:

-   -   molecular weight 82 Kda;    -   specific activity between 1000 and 1800 nkat/mg;    -   purity between 98.0 and 100%;    -   absence of microbial and protein contaminants, specifically        absence of endotoxins and DNA;    -   stability at a pH of between 5.5 and 11;    -   stability in aqueous solution at a T ranging between 4° and 40°        C., particularly stable at 37° C.;    -   stability in aqueous solution at 4° C. for 30 days;    -   stability in aqueous solution at a T ranging between −20° C. and        −80° C. for 24-48 months;    -   lyophilisability to obtain a stable freeze-dried powder.

It is preferably also characterised by

-   -   N-terminal sequence:        H₂N-Thr-Ala-Cys-Asp-Leu-Glu-Ala-Leu-Val-Thr-Glu-Ser-Ser-Asn-Gln        (SEQ ID NO:3);    -   inhibition by Ag and Cu salts and the chelating agent EDTA;    -   storability at temperatures ranging between −20 and −80° C.,        i.e. in frozen form, without significant loss of enzyme activity        (5-15%).

The collagenase production and purification process according to theinvention comprises the following stages:

Stage A: Inoculation of Vibrio alginolyticus chemovar. iophagus into anErlenmeyer flask and fermentation with culture broth of non-bovineanimal origin;

Stage B: Clarification of the fermented broth thus obtained bytangential flow ultrafiltration (TFF1) with 100-500 kD Molecular WeightCut-Off (MWCO) cassettes, preferably 300 kD;

Stage C: Dialysis and concentration of the clarified medium obtained instage B, by tangential flow ultrafiltration (TFF2) with 5-30 kD MWCOcassettes, preferably 10 kD MWCO;

Stage D: Purification of the solution containing collagenase obtained inStage C, by anion-exchange resin carrying weak basic groups, at a pH ofbetween 6.9 and 7.4, preferably at a pH of 7.1;

Stage E: Dialysis and concentration of the fractions collected whichhave collagenolytic activity, originating from Stage D, by tangentialflow ultrafiltration (TFF3) with 10-50 kD MWCO cassettes, preferably 30kD MWCO;

Stage F: Purification of the solution thus obtained, by anion-exchangeresin carrying strong basic groups, at a pH of between 6.9 and 7.4,preferably at a pH of 7.1;

Stage G: Diafiltration and concentration of the fractions withcollagenolytic activity ≥95% originating from stage F, by tangentialflow ultrafiltration (TFF4) with 10-50 kD MWCO cassettes, preferably 30kD MWCO;

Stage H: Filtration of the solution containing collagenase thusobtained, through an 0.2 μm absolute filter, and storage at atemperature of between −20° and −80° C.

Tests with the materials and methods described in the “Test Methods”paragraph below are conducted at the end of each stage.

Stage A: is a batch fermentation process; the inoculum is prepared in anErlenmeyer flask containing a culture broth formed by a peptone ofnon-bovine animal origin, such as porcine origin, or a mixture ofpeptones of non-bovine animal and plant origin, NaCl, CaCl2 and TRIS(tris-hydroxymethyl-aminomethane), at a pH of between 6.9 and 7.4,preferably 7.1. When the optical density measured at 600 nm (OD_(600nm))reaches a value of between 1 and 4, the inoculum is ready for transferto the fermenter.

The medium used for fermentation is the same as used to prepare theinoculum, with the addition of a small amount of antifoam to preventfoam formation due to aeration and stirring.

One fermentation lasts for 14-20 hours, normally 16 hours. Duringfermentation, samples are taken by a sterile procedure to check thepurity, OD_(600nm), enzyme activity and pH.

When the enzyme activity is ≥25,000 nkat/litre, fermentation isterminated and CaCl₂ is added to stabilise the enzyme. The temperatureis reduced to approx. 8° C., and the mixture is left under stirring.

The following control tests are performed on the solution obtained atstage A: OD_(600nm), pH; enzyme activity; protein concentration;SDS-PAGE.

The fermentation broth originating from stage A presents, in addition tothe collagenase of interest, other proteases produced by the bacteriumduring fermentation (mainly serine protease), protein aggregates withhigh molecular weight and residues of the medium.

Stage B: the fermented broth originating from stage A is clarified bytangential flow ultrafiltration (TFF1) with 100-500 kD MWCO cassettes,preferably 300 kD; this eliminates the microbial cells and proteinaggregates with high molecular weight.

The following control tests are performed on the solution obtained atstage B: pH; enzyme activity; protein concentration; SDS-PAGE; caseinaseassay.

Stage C: the clarified medium originating from stage B is concentratedand dialysed by ultrafiltration with 5-30 kD MWCO cassettes, preferably10 kD MWCO, approx. 15-25 times. The solution obtained at stage Ctypically presents enzyme activity of 500-700 nkat/ml, and is stable for12 months at −20° C. The purpose of tangential flow filtration with 5-30kD MWCO cassettes is to considerably reduce the volume and replace theculture medium with the 25 mM TRIS-HCl, 10 mM CaCl₂, pH 7.1 buffer,which stabilises the collagenase and is suitable for the subsequentpurification process.

The following control tests are performed on the solution obtained atstage C: pH; enzyme activity; protein concentration; SDS-PAGE.

Stage D: the solution containing the collagenase deriving from stage Cundergoes the first chromatographic purification with anion-exchangeresin carrying diethylaminoalkyl groups, preferably diethylaminoethyl,such as DE-52 (diethylaminoethyl cellulose DEAE Whatman). These resinscarry weak basic groups and therefore have a degree of ionisationdependent on the pH, with a narrow range of pHs between 6.9 and 7.4,preferably 7.1.

The solution containing the collagenase deriving from stage C is thenloaded into a column (Pall Chromatography Column Resolute Mod.400-V-EP7040) packed with DE-52 resin. The chromatography runs aremonitored by a UV-vis detector at 280 nm.

Before the column is loaded it is equilibrated with the same buffer,hereinafter called “equilibration buffer” (25 mM TRIS-HCl, 10 mM CaCl₂,pH 7.1), in which the collagenase was dialysed during stage C.

After loading, the resin with the bonded collagenase is eluted with saidequilibration buffer, to eliminate the proteins not bonded to the resin.A second wash with a buffer with greater conductivity, hereinaftercalled “washing buffer” (300 mM TRIS-HCl and 10 mM CaCl₂ at pH 7.1), isperformed to eliminate impurities with low molecular weight. Thecollagenase bonded to the resin is eluted by further increasing theconductivity with a third buffer called the “elution buffer” (300 mMTRIS-HCl, 700 mM NaCl and 10 mM CaCl₂ at pH 7.1).

Stage E: the fractions collected during elution which presentcollagenolytic activity and exhibit good purity in SDS-PAGE are combinedand transferred to the ultrafiltration system with 10-50 kD MWCOcassettes, preferably 30 kD, to be concentrated and dialysed in a bufferthat stabilises the collagenase and is suitable for the subsequentpurification process.

The following control tests are performed on the solution obtained atStage E: pH; enzyme activity; protein concentration; SDS-PAGE; caseinaseassay.

Stage F: the solution originating from Stage E passes to the secondpurification stage with anion-exchange chromatography using a resincarrying strong basic exchanger groups formed by quaternary ammonium,such as Source™15Q (GE Healthcare). The chromatography runs aremonitored by a UV-vis detector at 280 nm.

Before loading, the column (Millipore GS 70-550) packed with Source™15Qresin is equilibrated with the same buffer in which the collagenase wasdialysed during stage E (equilibration buffer).

The solution containing the collagenase originating from stage E isloaded into the column, and the resin with the bonded collagenase iseluted with the equilibration buffer to eliminate the unbonded proteins.The collagenase bonded to the resin is eluted by further increasing theconductivity with a second buffer (elution buffer 2-300 mM TRIS-HCl and10 mM CaCl₂ at pH 7.1).

Stage G: the fractions collected during elution which presentcollagenolytic activity and exhibit a purity of ≥95% in SDS-PAGE arecombined and transferred to the ultrafiltration system with 10-50 kDMWCO cassettes, preferably 30 kD, to be concentrated and dialysed in abuffer that stabilises the collagenase.

The following control tests are performed on the solution obtained atStage G: pH; enzyme activity; protein concentration; SDS-PAGE.

Stage H: the collagenase solution originating from stage G is diluted instabilising buffer and filtered through an 0.2 μm absolute filter. Thesterile solution thus obtained is the final product of the purificationprocess and is analysed for the following characteristics:

-   -   protein concentration    -   enzyme activity    -   pH    -   caseinase assay    -   SDS purity    -   dimer and high molecular weight analysis with UPLC SEC    -   endotoxins    -   sterility tests

Analysis Methods

Spectrophotometric determination of enzyme activity of collagenase(Wünsch, E., & Heidrich, H. G., modified)

The present method allows the activity of the collagenase present inaqueous solutions to be determined. The activity is expressed in katals,defined as the quantity of enzyme that catalyses the transformation of 1mole of substrate in 1 sec under the conditions specified by the method.The quantity of enzyme that catalyses the transformation of thesubstrate in a pre-set time at 37° C. and pH 7.1, related to thequantity of solution analysed, is expressed in nkat/ml. The principle ofthe method is based on the reaction between collagenase and thesynthetic substrate PZ-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine(where PZ=4-phenylazobenzyloxycarbonyl) specific for collagenase. Afterreacting with collagenase the synthetic substrate is cleaved into 2fragments, PZ-L-prolyl-L-leucyl and glycyl-L-prolyl-D-arginine. Thesecond fragment is colourless, while the first is a chromophore and canbe determined spectrophotometrically after extraction with an organicsolution of ethyl acetate acidified with citric acid. The absorbance ofthe fragment at 320 nm is proportional to the enzyme activity.

To perform the enzymatic assay it is necessary to prepare a series ofdilutions of the sample so as to have an enzymatic concentration thatfalls into the linearity range of the method.

The sample to be analysed is diluted in 25 mM Tris, 10 mM CaCl₂, pH 7.1buffer; 0.5 ml of this buffered solution is reacted at 37° C. for 15 minwith 2 ml of a 1.23 mM solution of synthetic substrate. At the end ofthe enzymatic reaction, 0.5 ml of the reaction mixture is extracted withorganic phase with a mixture of 5:1 ethyl acetate and 0.5% citric acid,pH 3.5. The organic phase is removed and dehydrated by adding 300 mg ofanhydrous sodium sulphate. The dehydrated organic phase is analysedspectrophotometrically at 320 nm against ethyl acetate. The results ofthe enzyme activity, expressed in nkat/ml, are calculated with thefollowing formula:

${{Activity}\mspace{14mu}{{nkat}/{ml}}} = {\frac{\begin{matrix}{( {{{sample}\mspace{14mu}{Abs}_{320}} - {blankAbs}_{320}} ) \times} \\{{Std}.\;{Conc}.\mspace{11mu}( {{\mu moles}/{ml}} )}\end{matrix}}{{{Std}\mspace{20mu}{Abs}_{320}} - {{blank}\mspace{14mu}{Abs}_{320}}} \times \frac{50 \times 1000}{900} \times {fd}}$

1000=conversion factor from μmol to nmol

900=seconds in 15 minutes

fd=conversion factor for the initial dilution of the collagenasesolution

50=conversion factor for dilution of the sample (0.5 ml and dilute to2.5 ml. 0.5 ml and dilute to 5 ml)

Std Conc. (μmol/ml)=0.02=0.4 ml of the 250 μM solution diluted to 5 ml.

The blank is given by the same enzymatic reaction as collagenase whereinthe solution containing the enzyme is replaced with the reference buffer(25 mM Tris, 10 mM CaCl₂, pH 7.1).

The Standard is an 0.2504 mM solution of reaction fragmentPZ-L-prolyl-L-leucine in ethyl acetate;

0.4 ml of this solution is added to a solution consisting of 4.6 mlethyl acetate and 1 ml 0.5% citric acid, pH 3.5. The organic phase isremoved and dehydrated by adding 300 mg of anhydrous sodium sulphate.The dehydrated organic phase is analysed spectrophotometrically at 320nm against ethyl acetate.

Determination of Protein Concentration by the Lowly Method

The protein concentration of solutions containing collagenase isdetermined by the Lowly method according to the following references:

-   -   1. European Pharmacopoeia 5.0, Total Protein, Chapter 2.5.33;    -   2. Lowry, O. H. et al., 1951, “Protein measurement with the        Folin phenol reagent”, J. Biol. Chem., 193, 265-275.

UPLC Size Exclusion

UPLC size exclusion analysis is used to determine the dimers andmolecules with high molecular weight (defined as impurities) and/orcollagenase degradation products. The instrument used is an Acquity UPLCH-Class with PDA eλ detector equipped with an Acquity UPLC BEH 200 SECcolumn. The analysis is conducted in isocratic mode using a pH 6.4-6.7phosphate buffer formulated as follows: Na₂PO₄ 8.9 g/1, NaH₂PO₄ 6.9 g/land NaCl 8.76 g/l. The buffer is filtered through 0.2 μm absolutefilters before use. 1.5-4 μg of protein is injected into a 5 μl volumefor each test.

SDS-PAGE Electrophoresis

Electrophoretic analysis on 10% polyacrylamide gel in the presence ofsodium dodecyl sulphate (SDS) is conducted according to the Laemmlimethod (Laemmli, U. K., 1970, “Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4”, Nature, 227, 680-685.

Determination of Caseinase

Caseinase determination is used as the method for assaying the aspecificproteases present as impurities in the collagenase solution. Theanalysis method is performed according to Anson, M. L. (1938) J. Gen.Physiol. 22, 79-89, and Folin, O. and Ciocalteu, V. (1927) J. Biol.Chem. 73, 627-650. The caseinase is determined using casein assubstrate. The reaction can be schematically illustrated as follows:

${{Casein} + {H_{2}O}}\overset{\mspace{20mu}{Protease}\mspace{25mu}}{arrow}{{Amino}\mspace{14mu}{acids}}$

The quantity of tyrosine produced by the reaction is determinedcolorimetrically by exploiting the reaction with Folin-Ciocalteureagent, which has the property of oxidising tyrosine in an alkalineenvironment so that it develops a blue colour. Briefly, 1 ml of solutionto be analysed is added to 5 ml of 0.65% (W/V) casein solution and leftto react for 30 minutes at 37° C. 0.5 ml of trichloroacetic acid (TCA)is added at the end of incubation, and the sample is filtered through0.45 μm filters after 2 minutes. The filtrate is collected; 5 ml of 0.5M Na₂CO₃ and 1 ml of 0.5 N Folin-Ciocalteu are added and the mixture isleft to react for 30 minutes at 37° C. At the end of the reaction thesample is further filtered through 0.45 μm filters and the filtrate isanalysed with the spectrophotometer, measuring the absorbance at awavelength of 660 nm. At the same time the blank and the calibrationline are prepared with a solution of 1.1 mM L-tyrosine as standard.

The results are calculated with the following formula:

$\frac{Unit}{ml} = {\frac{\frac{( {O.D._{sample}{- {O.D._{blank}}}} ) - c}{m} \times 10 \times 6,5 \times 8}{1 \times 2} \times {fd}}$

c=known term

m=angular coefficient

10=conversion factor from 30 minutes to 5 hours

6.5=total volume in ml of stop solution

8=total volume of colorimetric solution

1=ml of collagenase sample

2=ml of sample used for colour development

fd=dilution factor

Measurement of Optical Density OD_(600nm)

This method allows the growth of the cells during the various stages ofthe production process to be evaluated, from the 5-litre Erlenmeyerflask to the 1000-litre bioreactor. To perform the OD_(600nm)measurement, 1 ml of cell suspension is taken up and centrifuged at12000 rcf for 5 min, the supernatant is eliminated and the cellsresuspended in 1 ml of distilled H₂O, after which the absorbance is readat 600 nm.

Endotoxin Assay

The endotoxin assay in the final collagenase solution is performed asdescribed in the European Pharmacopoeia, Endotoxin Test, Chapter 2.6.14.

Determination of Enzyme Activity of Collagenase by UPLC

The present method allows the activity of collagenase to be quantifiedby UPLC analysis. The method is based on quantification of the fragmentproduced by the enzymatic reaction (according to the Wunsch method)against the external standard.

The quantity of enzyme that catalyses the transformation of thesubstrate in a pre-set time at 37° C. and pH 7.1, related to thequantity of solution analysed, is expressed in nkat/ml. The activity isexpressed in katals, defined as the quantity of enzyme that catalysesthe transformation of 1 mole of substrate in 1 sec under the conditionsspecified by the method.

The range of use of the method in linear conditions extends to approx.1.2 nkat/ml, as the maximum activity of collagenase in solutionanalysable according to the methodology described, with no need toperform “dilutions”.

The aqueous solution of collagenase is reacted with the syntheticsubstrate PZ-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine (wherePZ=4phenylazobenzyloxycarbonyl). Two fragments are released undercontrolled conditions (pH, temperature, time): PZ-L-prolyl-L-leucine;glycyl-L-prolyl-D-arginine. The second fragment is determined by UPLC.

The operating parameters of the UPLC system are:

-   -   flow rate: 0.919 ml/min    -   duration of run: 3 minutes    -   wavelength: 320 nm    -   injection volume: 1.4 μl    -   column temperature: 25° C.

time (minutes) Citric acid 0.5% w/v pH 3.5 Acetonitrile 0 50 50 1.17 5050 0.34 10 90 1.13 10 90

Under these conditions the substrate elutes after approximately 0.25minutes and the fragment after approx. 0.44 minutes

The determination of the enzyme activity consists of the followingsteps:

I. Preparation of a 25 mM TRIS-HCl, 10 mM CaCl₂, pH 7.1 buffer solution(Reagent A).

II. Preparation of an 0.5% citric acid solution (Reagent B).

III. Preparation of a 1.23 mM substrate solution (Reagent C).

IV. Preparation of the solution of PZ-L-prolyl-L-leucine 200 μM inacetonitrile (reagent E).

V. Preparation of solution of collagenase to be analysed (solution D).The enzymatic solution is diluted in a volumetric flask with the buffersolution (Reagent A), to obtain a solution with activity less than 1.2nkat/ml.

VI. Enzymatic reaction. 2 ml of reagent C and 0.5 ml of solution D arepipetted into a screw-cap test tube using a glass pipette. The mixtureis left to react for 15 min at 37° C. in a thermostatic bath. At the endof the reaction, this is called solution Y. A “blank”, W, is prepared inparallel as follows: 2 ml of reagent C and 0.5 ml of buffer solution(reagent A). The mixture is left to react for 15 min at 37° C. in athermostatic bath.

VII. The samples are prepared by taking up 0.5 ml of solution Y andintroducing it into a 10 ml flask containing 2.0 ml of 0.5% citric acid,and made up to volume with acetonitrile.

VIII. The reference solution is prepared by introducing 0.5 ml of blankW, 2.0 ml of citric acid and 1.5 ml of reagent E into a 10 ml flask, andmaking it up to volume with acetonitrile.

IX. 1.4 μl of the reference solution followed by 1.4 μl of the solutionto be analysed are injected.

The results of the enzyme activity expressed in nkat/ml are calculatedwith the following formula:

${{Activity}\mspace{14mu}{{nkat}/{ml}}} = {\frac{{Sample}\mspace{14mu}{Area} \times {Standard}\mspace{14mu}{{Conc}.\mspace{14mu}( {{\mu moles}/{ml}} )}}{{Standard}\mspace{14mu}{Area}} \times \frac{1000 \times 100}{900} \times {fd}}$

Calculation of enzyme activity=nanomoles per second per ml of solution(nkat/ml), where:

1000=conversion factor from micromoles to nanomoles

100=conversion factor for dilution of sample (0.5 ml to 2.5 ml. 0.5 mlto 10 ml)

900=seconds in 15 minutes

Standard Conc. (μmoles/ml)=0.03 (1.5 ml of the 200 μM solution to 10 ml)

fd=dilution factor of the collagenase solution used to prepare solutionD

Characterisation of Protein

Determination of N-Terminal Sequence

The N-terminal amino-acid sequence of the collagenase originating fromVibrio alginolyticus chemovar. Iophagus was determined by the Edmandegradation method using a liquid-phase automated protein sequencer(ABI-Perkin Elmer mod. 477°). The sequence obtained was verified bybioinformatic analysis, running BLAST (Basic Logical Alignment SearchTool) searches of the sequence against the entire GENBANK database.

Peptide Mapping analysis

To analyse the peptide map, the collagenase is first reduced with DTT,alkylated with iodoacetamide and then desalted using a PD10 column,eluting with 1% acetic acid. The eluate is subjected to trypticdigestion, and the fragments are analysed with MALDI-MS. The globalspectrum is reported in (FIG. 1 : tryptic digest analysis); this enabledus to establish that over 90% of the amino-acid sequence is identical tothe sequence deposited in the database corresponding to the collagenaseproduced from Vibrio alginolyticus chemovar Iophagus.

Circular Dichroism

The far-UV circular dichroism (FUV-CD) spectra were recorded on a Jascospectrum polarimeter, model J-810, connected to a bath thermostated at25° C. The spectra were recorded using an 0.1 cm cuvette at the speed of20 nm/min, with a response every 8 sec, for an average of twoaccumulations. Each sample was tested in duplicate.

The CD signal was expressed as ellipticity per mean residue, calculatedwith the formula [θ]=θobs×MRW/(10×l×c), wherein θobs is the ellipticityobserved in mdeg, “MRW” is the mean molecular weight per residue, “I” isthe optical path in cm and “c” is the concentration in mg/ml. Thesamples were analysed at the concentration of 0.2 mg/ml.

Example 1: Production and Purification of Collagenase from Vibrioalginolyticus Chemovar. Iophagus, in an 800 L Fermentation

(FIG. 2 : Flow chart of production and purification process)

Fermentation

Fermentation is divided into 2 stages:

Stage 1: Preparation of inoculum

Stage 2: Fermentation

Stage 1: The culture medium is liquid, and consists of a solution of1.21 g/l TRIS, 23.4 g/l NaCl, 0.29 g/l CaCl₂ and 15 g/l peptone ofnon-bovine animal origin (porcine, or a mixture with peptones of porcineand plant origin) dissolved in distilled water (Millipore milliQ). ThepH of the medium is adjusted to 7.1 with HCl and sterilised in theautoclave at a temperature of ≥122° C. for 30 min. A 1.5 ml ampoule ofVibrio alginolyticus chemovar. Iophagus (WCB-Working Cell Bank) isinoculated into a 5 L Erlenmeyer flask containing 2 L of culture mediumand incubated at 30° C. with stirring at approx. 150 rpm, for a timeranging between 8 and 16 hours. When the OD_(600nm) reaches a value ofbetween 1 and 4, the inoculum is ready to be transferred to thefermenter.

Stage 2: the culture medium is the same as used for Stage 1, with theaddition of 0.25 g/l of Sigma 204 antifoam, sterilised at ≥122° C. for30 min in the fermenter. The 2 L of inoculum is transferred by a sterileprocedure to the fermenter containing 800 L of fermentation broth, andthe fermentation parameters are set as follows:

-   -   temperature 30° C.±1° C.,    -   stirring 50-150 rpm,    -   air 10-80 Nm³/h,    -   dissolved oxygen >50%,    -   pH 7.1±0.1,    -   pressure 0-0.4 bar.

A typical fermentation lasts for 14-20 hours, normally 16 hours, whichcorresponds to the highest level of collagenase enzyme activitythroughout the fermentation process. During fermentation, samples aretaken by a sterile procedure to check the purity of the culture, theOD_(600nm) and enzyme activity. The activity of the collagenase isdetermined spectrophotometrically by the modified Wünsch-Heidrich method(Wünsch, E. & Heidrich, H. G. 1963).

When the enzyme activity is ≥25,000 nkat/litre, fermentation isterminated and CaCl₂ is added, up to a final concentration of 1.47 g/l,to stabilise the enzyme. The temperature is lowered to approx. 8° C.,and the mixture is left under stirring for approx. 10-30 minutes.Typically, a fermented broth has the following characteristics:

1) Enzyme activity between 25,000 and 50,000 nkat/litre

2) OD_(600nm) between 5 and 8

3) Absence of microbial contaminants

Clarification: TFF1 Ultrafiltration with 300 kD MWCO (Molecular WeightCut-Off) Cassettes

The fermentation broth, approx. 800 litres, is transferred to theultrafiltration system containing SARTOCON II holder (Sartorius) wherefive 300 kD MWCO cassettes (Code 3021467907E-SG) are housed, each with afiltration area of 0.5 m². The filtration membrane is made of modifiedPolyEtherSulphone (PES), the main characteristic of which is a lowaffinity for proteins, allowing >80% recovery and clarification of themedium with low loss of collagenase.

The objective of ultrafiltration with 300 kD cassettes is to remove themicrobial mass from the fermentation broth and eliminate a proteinaggregate of approx. 320 Kda.

Dialysis and Concentration: TFF2 Ultrafiltration with 10 kD MWCOCassettes

After clarification the medium is concentrated and dialysed in the PALLMod UF-A-P0971 ultrafiltration system in which six 10 kD MWCO cassettes(Code 34293012R), each with a filtration area of 0.5 m², are housed. Thefiltration membrane is made of modified PolyEtherSulphone (PES), themain characteristic of which is a low affinity for proteins,allowing >80% recovery. Ultrafiltration with 10 kD cassettes allows thevolume to be reduced 15-25 times, low molecular weight contaminants tobe eliminated, and the culture medium replaced with 25 mM TRIS, 10 mMCaCl₂, pH 7.1 buffer, which is suitable for the subsequent purificationprocess. After the ultrafiltrations, the following analyses areperformed:

1) Enzyme activity

2) Protein assay

3) SDS-PAGE (FIG. 3 : SDS-PAGE of the 300K and 10K ultrafiltrationstages)

Legenda of FIG. 3

Lane 1 High Range SigmaMarker™

Lane 2 10 μl fermentation broth

Lane 3 20 μl fermentation broth

Lane 4 10 μl TFF 300K filtrate

Lane 5 20 μl TFF 300K filtrate

Lane 6 2 μl TFF 10K retentate

Lane 7 5 μl TFF 10K retentate

Lane 8 16 μl TFF 10K retentate

Lane 9 2 μg FIDIA collagenase

Weak Anionic Chromatography (Whatman DE-52)

The solution containing the collagenase deriving from theultrafiltration processes is the starting material for the firstpurification by weak anion-exchange chromatography using DE-52 resin(Whatman DEAE: diethylaminoethyl cellulose).

The chromatography runs are monitored by a UV-vis detector at 280 nm.Typically, 10-25 litres of the solution containing collagenase with aprotein concentration of 0.8-1.2 g/l are loaded into the column (PallChromatography Column Resolute Mod. 400-V-EP7040) packed with 10 Kg ofDE-52 resin. The quantity of total proteins loaded into the column is0.8-3 g/Kg of resin. The resin is equilibrated with 10 column volumes(BV) of 25 mM TRIS-HCl and 10 mM CaCl₂ at pH 7.1, hereinafter called“equilibration buffer”.

After loading, the resin with the bonded collagenase is eluted with 2-4BV, generally 2, of equilibration buffer to eliminate the proteins notbonded to the resin and restore the conductivity values to those priorto loading of the sample.

To eliminate the low molecular weight impurities, a further elution isperformed with 3-5 BV, generally 4, of a buffer formulated as follows:300 mM TRIS-HCl and 10 mM CaCl₂ at pH 7.1 (washing buffer). Thecollagenase bonded to the resin is eluted by increasing the conductivitywith 3-5 BV, generally 4, of a third buffer composed as follows: 300 mMTRIS-HCl, 700 mM NCl and 10 mM CaCl₂ at pH 7.1 (elution buffer).

The fraction collected during elution with the elution buffer issubjected to an enzyme activity assay and analysed with SDS-PAGE.

A typical chromatographic profile presents three distinct peaks, asshown in FIG. 4 (typical chromatogram of DE-52):

-   -   the first (I) (peak eluted with equilibration buffer)        corresponds to the unbonded proteins, which presents no enzyme        activity.    -   the second peak (II) corresponds to the elution with the washing        buffer, which presents collagenolytic activity but is discarded        because numerous impurities are also present.    -   the third peak (III) corresponds to elution with the elution        buffer, and is the peak with the greatest enzyme activity and        highest purity.

Typically, the peak containing collagenase has a volume of 18-22 litres,and is analysed for the following characteristics:

1) Enzyme activity

2) Protein assay

3) SDS-PAGE (FIG. 5 )

Legenda of FIG. 5

Lane 1 5 μl of sample after TFF2

Lane 2 20 μl unbonded

Lane 3 10 μl peak I fraction 1

Lane 4 10 μl peak I fraction 2

Lane 5 10 μl peak II fraction 1

Lane 6 10 μl peak II fraction 2

Lane 7 10 μl peak III

Dialysis and Concentration: TFF3 Ultrafiltration with 30 kD MWCOCassettes

The fraction corresponding to the third chromatographic peak isconcentrated and dialysed in the Cogent™ (Millipore) ultrafiltrationsystem in which two 30 k MWCO cassettes (Code 31158044R), each with an0.1 m² filtration area, are housed. The filtration membrane is made ofPolyEtherSulphone (PES) whose main characteristic is a low affinity forproteins, allowing recovery >95%. Ultrafiltration with 30 kD cassettesallows the volume to be reduced 3-6 times and the elution buffer fromDE-52 to be replaced with 25 mM TRIS, 10 mM CaCl₂, pH 7.1 buffer, whichis suitable for the subsequent purification process. The followingcontrols are performed on the ultrafiltrate:

1) Enzyme activity

2) Protein assay

Strong Anionic Chromatography (Source™15Q GE Healthcare).

The collagenase solution originating from TFF3 is the starting materialfor the second purification by strong anion-exchange chromatographyusing Source™15Q resin (GE Healthcare). The chromatography runs aremonitored by a UV-vis detector at 280 nm. Typically, 3-6 litres ofsolution containing collagenase with a protein concentration of 0.8-1.2mg/ml are loaded into a column (Millipore GBP 70-550) packed with100-200 ml of Source™15Q resin; the quantity of total proteins loadedinto the column amounts to 24-36 mg/ml of resin. Before loading, theresin is equilibrated with 2 column volumes (BV) of 10 mM TRIS-HCl and10 mM CaCl₂₅ at pH 7.1, called the “equilibration buffer”.

After loading, the resin with the bonded collagenase is eluted with 5-7BV of equilibration buffer to eliminate the unbonded proteins andrestore the conductivity to the initial values. The collagenase bondedto the resin is eluted with 5-10 BV of 300 mM Tris-HCl and 10 mM CaCl₂at pH 7.1 (elution buffer). The fraction containing collagenase iseluted in a single peak, typically of 1-2 litres, which can be seen inthe chromatogram shown in FIG. 6 (Typical chromatogram of Source™15Q).

The fraction containing the collagenase collected during elution issubjected to the following controls:

1) Enzyme activity

2) Protein assay

3) SDS-PAGE (FIG. 7 : SDS-PAGE of Source Fractions™15Q)

Legenda of FIG. 7 :

Lane 1 20 μl unbonded conc. 20X Lane 2 10 μl Fraction 1 Lane 3 10 μlFraction 2 Lane 4 LMW

Dialysis and Concentration: TFF4 Ultrafiltration with 30 kD MWCOCassettes

The fraction containing collagenase is transferred to the Cogent™(Millipore) ultrafiltration system in which two 30 kD MWCO cassetteswith PolyEtherSulphone (PES) membrane, each with an 0.1 m² filtrationarea, are housed. This ultrafiltration step allows the sample to bedialysed and concentrated against the 25 mM TRIS-HCl and 10 mM CaCl₂ pH7.1 buffer.

Filtration and Storage

The solution originating from TFF4 is filtered through 0.2 μmPolyEtherSulphone (PES) absolute filters for final sterilisation of theproduct. The end product is stored in specific containers at −20° C.

The collagenase solution thus obtained is analysed for the followingcharacteristics: protein concentration; enzyme activity; pH; aspecificprotease assay; SDS-PAGE purity (FIG. 8 : SDS-PAGE of final product);UPLC SEC purity (dimer and high molecular weight analysis); LAL Test fordetermination of endotoxins (according to the European Pharmacopoeia,the product must contain no more than 5 IU/kg/hour).

Legenda of FIG. 8 :

Lane 1 HMW Lane 2 Fidia Collagenase 0.5 μg/lane Lane 3 Fidia Collagenase1 μg/lane Lane 4 Fidia Collagenase 2 μg/lane

The specific characteristics are summarised in Table below.

TABLE TEST SPECIFICATIONS Identification: positive UPLC size exclusionAppearance Clear, colourless solution in 25 mM TRIS-HCl, 10 mM CaCl₂, pH7.1 pH of solution 7.1 ± 0.2 Protein concentration ≤1.0 mg/ml ofsolution Specific activity 1000-1800 nkat/mg Caseinase activity <1.0U/ml Purity in UPLC 98.0-100% Identification Molecular weight 82 KDaPurity in SDS-PAGE 98.0-100% Endotoxins Absent or conforming to EuropeanPharmacopoeia

The collagenase produced and purified according to the present inventionhas been tested by the Applicant to verify the followingcharacteristics, with the results set out below:

-   -   pure: has a purity of between 98 and 100%;    -   free of microbial and protein contaminants (endotoxins, DNA);    -   stable at a pH of between 5.5 and 11 (established by enzymatic        test)    -   stable in aqueous solution, for example comprising 25 mM        TRIS-HCl, 10 mM CaCl₂, pH 7.1, at a T of between 4° and 40° C.,        particularly stable a 37° C.    -   stable in aqueous solution at 4° C. for 30 days;    -   stable in aqueous solution at a T of between −20° C. and −80° C.        for 24-48 months    -   can be freeze-dried with suitable excipients (described below)        to obtain a stable lyophilic powder.

The lyophilisate of collagenase, which is a further subject of thepresent invention, is prepared with excipients particularly suitable tomaintain its stability, and can preferably contain:

-   -   maltose: 95-96%, preferably 95.75%;    -   salts (such as TRIS-HCl+CaCl₂): 1.0-1.5%, preferably 1.3%;    -   collagenase: 2.5-3.5%, preferably 2.95%, to obtain a        freeze-dried powder with enzyme activity between 7-20 nkat/mg of        powder

The quantities indicated above for collagenase lyophilisate arepercentages by weight compared with the total weight of thelyophilisate.

The collagenase obtained according to the production and purificationprocess is therefore suitable for the preparation of pharmaceuticalcompositions of various types, and is designed, as stated, for the usein the treatment of disorders characterised by collagen accumulation ordermocosmetic treatment of blemishes/imperfections that benefit from areduction in local collagen accumulations. The most frequentapplications relate to topical and/or local treatment of burns ofdifferent degrees, scalds, bedsores, vascular ulcers and diabeticulcers; in these cases, collagenase effectively eliminates the eschar,thus allowing the viable underlying cells to activate for the purpose ofthe repair process. Other therapeutic applications relate to cellulitis,post-surgical adhesions, hypertrophic scars and keloids; in thesesituations, abnormal collagen production has adversely affected thenormal repair process, and they can benefit from lysis of theirregularly accumulated collagen.

Other disorders treatable with collagenase are adhesive capsulitis(frozen shoulder), Dupuytren's contracture and Peyronie's disease.

Topical or systemic application, in particular injectable application,may be required, depending on the disorder.

As regards topical applications, the surprising stability in aqueoussolution of the enzyme claimed herein allows its formulation inhydrophilic carriers; in particular, it has been found that collagenasecan be formulated with particular polymers which hydrate in contact withthe exudate of the lesion on which it is placed, giving rise to a gel.In this situation, the enzyme is released gradually and in largerquantities than obtained with the ordinary lipophilic carriers used todate, producing a better therapeutic effect; the number of dailyapplications can also be reduced, thus improving the patient'scompliance. The topical pharmaceutical form preferred in the ambit ofthe present invention is dusting powder, the industrial preparation ofwhich does not require any particular processes and which, in the formof a pharmaceutical preparation, can easily be measured, correctlydeposited within the edges of the lesion, and stored for long periods.The hydrophilic polymer used must be able to absorb the fluid producedby the wound rapidly, producing an adherent gel material. Said gel mustobviously have viscosity characteristics which allow its residence onthe wound bed; an excessively solid gel tends to vitrify, while anexcessively liquid gel slides off the wound bed, carrying the activeingredient with it. Moreover, as the preparation must be sterile, it isessential for the polymer selected to maintain its rheologicalproperties after hydration, even when previously sterilised by commonmeans (usually γ rays). To identify the most suitable polymer, theApplicant has conducted a series of tests on numerous polymers (starchand derivatives, alginates, cellulose derivatives, polyvinyl alcoholsand derivatives, gums and pectins), as briefly described herein. Acontinuous film of saline solution 2.5 mm thick was created in a glassPetri dish with a diameter of 5 cm, on which the powdered polymer to beexamined (approx. 1 g) was sprinkled from a steel sieve with pores offixed size. Further liquid was added as long as the polymer, convertedto a gel, was able to absorb it, maintaining a viscosity such that itdid not drip from the dish when held vertically. After the absorptionrate of the liquid and the transparency and consistency of the gelobtained were evaluated, it was decided that the most suitable polymerfor the purpose of the present invention is corn starch glycolate.

Corn starch glycolate is a fine white, flowable, very hygroscopicpowder, used in the manufacture of tablets and capsules due to itsdisintegrating properties, but has never been used by directapplication. Corn starch glycolate has a pH of between 5.5 and 7.5, i.e.similar to that of the tissue on which it is applied, and swells inwater to up to 300 times its volume.

In addition to the collagenase enzyme, the dusting powder can alsocontain a further active ingredient, which stimulates cell migration inorder to allow more rapid re-epithelialisation of the wound bed, andtherefore more rapid closing of the wound. Of the various possibleagents, hyaluronic acid is particularly suitable for these purposes; itis a heteropolysaccharide consisting of alternating residues ofD-glucuronic acid and N-acetyl-D-glucosamine. It is a straight-chainpolymer with a molecular weight ranging between 50,000 and 13×10⁶ Da,depending on the source from which it is obtained and the preparationmethods used. The HA used in the present invention can derive from anysource, such as extraction from cockscombs (EP 138572 B1), fermentation(from Streptococcus), or biosynthesis (from Bacillus), and have a meanmolecular weight of between 400 and 3×10⁶ Da, in particular between1×10⁵ Da and 1×10⁶ Da, and even more particularly between 200,000 and750,000 Da. Preferably, the HA used herein for topical applications hasa mean molecular weight (MW) of between 130 and 230 kDa, preferablybetween 145 and 210 kDa, and even more preferably between 160 and 200kDa; the latter will hereinafter be called HA with mean MW 200 kDa. HAwith a mean molecular weight of between 200 and 1800 kDa, preferablybetween 500 and 1300 kDa, and even more preferably between 750 and 1200kDa, is used for injectable applications. References to mean molecularweight refer to the weight-average MW, calculated by the intrinsicviscosity method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

HA is found in nature in pericellular gels, in the ground substance ofthe connective tissue of vertebrates (of which it is one of the maincomponents), in the synovial fluid of the joints, the vitreous humourand the umbilical cord.

It has been demonstrated that HA plays a crucial role in the tissuerepair process both in structural terms (in organising extracellularmatrix and regulating its hydration) and as a substance that stimulatesa wide range of processes in which it is directly and indirectlyinvolved (clot formation, phagocytic activity, fibroblast proliferation,neovascularisation, re-epithelialisation, etc.) (Weigel P. et al., JTheoretical Biol, 1986:219-234; Abatangelo G. et al., J Surg Res, 1983,35:410-416; Goa K. et al., Drugs, 1994, 47:536-566). The role of HA inthe preparations described herein is not only to promote wound-healing,but above all to prevent the collagenase that collects at the edges ofthe wound from damaging the living, healthy cells that reside there bypreventing their migration towards areas that need regeneration.Moreover, the presence of HA, which is also an absorbent polymer, aidsthe formation of the gel on the wound bed and improves the release, andtherefore the enzyme activity, of collagenase, as has been demonstratedand will be illustrated below.

The formulations identified can also include an agent that promotes theglide of the powders at the mixing stage; those most commonly usedinclude colloidal silicon dioxide, which can optionally be included inquantities ranging 0.1 and 3%, preferably between 0.2 and 1%, as knownto the skilled person.

In the ambit of the present invention the Applicant consequently intendsto claim pharmaceutical compositions for topical use in the form ofdusting powder, comprising:

-   -   collagenase obtained by the process described above, in a        quantity equivalent to an activity of between 2 and 8 nkat/g of        the finished product, preferably 5 nkat/g of the finished        product;    -   optionally, HA with a weight average molecular weight ranging        between 130 and 230 kDa, preferably between 145 and 210 kDa, and        even more preferably between 160 and 200 kDa, in a quantity        ranging between 0.1 and 5%, preferably between 0.2 and 2%;    -   optionally, colloidal silicon dioxide in a quantity of between        0.1 and 3%, preferably between 0.2 and 1%;    -   corn starch glycolate, in the quantity required to complete the        percentage composition.

The quantities indicated above for the dusting powder are percentages byweight compared with the total weight of the composition.

Some examples of preparation of the pharmaceutical compositionsdisclosed above will now be described by way of example but not oflimitation.

Example 2: Preparation of a Dusting Powder Containing Collagenase,Hyaluronic Acid (HA) and Corn Starch Glycolate (Formulation 1)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g 200 kDa mean MW HA 0.2 g Corn starchglycolate q.s. for 100 g

About half the quantity of gelling polymer is weighed and introducedinto the container; the collagenase and HA, previously micronised andsieved at 50μ, are weighed and introduced into the container. Finally,the remaining quantity of gelling polymer is weighed and introduced intothe container. The preparation is performed by direct mixing of thepowders in a parallelepipedal polyethylene vial closed with apolyethylene sub-cap and a polypropylene screw cap, with a sufficientcapacity to leave at least 40-50% of empty headspace. The vial, fixed inthe arm of a V mixer in an oblique position (45 degrees), rotatesobliquely about its minor axis at the speed of 50 rpm. Mixing proceedsuntil the mixture is homogenous.

Example 3: Preparation of a Dusting Powder Containing Collagenase,Hyaluronic Acid (HA), Corn Starch Glycolate and Glidant (Formulation 2)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g 200 kDa mean MW HA 0.2 g Colloidalsilicon dioxide 0.2 g Corn starch glycolate q.s. for 100 g

About half the quantity of gelling polymer is weighed and introducedinto the container; the collagenase and HA, previously micronised andsieved at 50μ, are weighed and introduced into the container. Finally,the remaining quantity of gelling polymer and the glidant are weighedand introduced into the container. The preparation is performed bydirect mixing of the powders in a parallelepipedal polyethylene vialclosed with a polyethylene sub-cap and a polypropylene screw cap, with asufficient capacity to leave at least 40-50% of empty headspace. Thevial, fixed in the arm of a V mixer in an oblique position (45 degrees),rotates obliquely around its minor axis at the speed of 50 rpm. Mixingproceeds until the mixture is homogenous.

Example 4: Preparation of a Dusting Powder Containing Collagenase,Hyaluronic Acid (HA), Corn Starch Glycolate and Glidant (Formulation 3)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g 200 kDa mean MW HA 0.2 g Colloidalsilicon dioxide 1 g Corn starch glycolate q.s. for 100 g

For preparation, see Example 2.

Example 5: Preparation of a Dusting Powder Containing Collagenase andCorn Starch Glycolate (Formulation 4)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g Corn starch glycolate q.s. for 100 g

Approx. half the quantity of gelling polymer is weighed and introducedinto the container; the collagenase is weighed and introduced into thecontainer. Finally, the remaining quantity of gelling polymer is weighedand introduced into the container. The preparation is performed bydirect mixing of the powders in a parallelepipedal polyethylene vialclosed with a polyethylene sub-cap and a polypropylene screw cap, with asufficient capacity to leave at least 40-50% of empty headspace. Thevial, fixed in the arm of a V mixer in an oblique position (45 degrees),rotates obliquely around its minor axis at the speed of 50 rpm. Mixingproceeds until the mixture is homogenous.

Example 6: Preparation of a Dusting Powder Containing Collagenase, CornStarch Glycolate and Glidant (Formulation 5)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g Colloidal silicon dioxide 0.2 g Cornstarch glycolate q.s. for 100 g

About half the quantity of gelling polymer is weighed and introducedinto the container; the collagenase is weighed and introduced into thecontainer. Finally, the remaining quantity of gelling polymer andglidant is weighed and introduced into the container. The preparation isperformed by direct mixing of the powders in a parallelepipedalpolyethylene vial closed with a polyethylene sub-cap and a polypropylenescrew cap, with a sufficient capacity to leave at least 40-50% of emptyheadspace. The vial, fixed in the arm of a V mixer in an obliqueposition (45 degrees), rotates obliquely around its minor axis at thespeed of 50 rpm. Mixing proceeds until the mixture is homogenous.

Example 7: Preparation of a Dusting Powder Containing Collagenase, CornStarch Glycolate and Glidant (Formulation 6)

The collagenase, in lyophilic form, is measured so that it correspondsto an activity of 5 nkat/g of finished product.

Collagenase equivalent to 5 nkat/g Colloidal silicon dioxide 1 g Cornstarch glycolate q.s. for 100 g

For preparation, see Example 5.

As stated, the pre- and post-sterilisation rheology of said dustingpowders, and their performance in terms of collagenase release, wereevaluated after hydration. This last test was conducted by comparisonwith a standard product (Bionect Start®) containing collagenase in alipophilic carrier.

Pre- and Post-Sterilisation Rheological Evaluation

The samples tested were prepared according to Examples 1 and 3, whichrepresent the most complex formulations of those identified.

1 g each of non-sterile Formulation 1 and Formulation 3 was hydratedwith 7 ml of saline solution; the same procedure was performed on 1 g ofthe same formulations pre-sterilised with γ rays (dose 25 kGray). Thegels thus obtained were analysed to evaluate viscous moduli G′ and G″with a HAAKE mod. Mars II viscometer equipped with plate-conemeasurement with 60 mm diameter and 1° angle; the measurement wasperformed at 20±0.5° C. under rotation control with a speed ramp withconstant acceleration from 0 to 5 sec−1 in 8 minutes; the interpolationwas performed at 1.0 sec−1. The results are illustrated in FIG. 11 (Pre-and post-sterilisation rheological evaluation): each formulationevidently retains its rheological properties practically unchanged, evenafter sterilisation. Formulation 1, without glidant, has slightlysuperior G′ and G″ moduli, but not to a statistically significantextent. This means that the glidant has no effect on the rheology, andwhether it is used or not therefore depends on the operating conditions.However, it must be emphasised that the polymer selected retains thecharacteristics of adherence and viscosity identified in the preliminarytests, which are necessary for the optimum release of the enzymecontained in the formulation.

Evaluation of Collagenolytic Activity

These tests test the behaviour of the formulations with and withouthyaluronic acid compared with a commercial reference product (BionectStart®) containing collagenase in a lipophilic carrier (ointment) andhyaluronic acid.

As the glidant has no effect on the enzyme activity of collagenase, itwas decided to test Formulations 1 (with HA) and 4 (without HA). Thedegradative activity of the enzyme is determined with a quantitativeassay on a standard commercial substrate (Collagenase Chromophoresubstrate kit—Fluka 27669). The method was suitably modified to allowtesting of the powdered formulation of the samples examined, andsimulation of the conditions to which said preparations will besubjected after application in vivo. The assay is based on hydrolysis ofthe specific collagenase substrate:4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg-OH (component A).Said substrate is degraded to Pz-Pro-Leu-OH (yellow-orange fragment) andH-Gly-Pro-D-Arg-OH in the presence of the collagenase enzyme. Thepeptide Pz-Pro-Leu-OH is soluble in organic solvents, and is extractedwith ethyl acetate from the mixture acidified with citric acid. Theexcess undegraded substrate remains in the acidified aqueous phase.Pz-Pro-Leu-OH in ethyl acetate is quantitatively determinedspectrophotometrically by reading at 320 nm.

Materials and Methods

Collagenase Chromophore substrate kit—Fluka 27669

Formulation 1

Formulation 4

Bionect Start®

Saline solution

TRIS Buffer

25 mM citric acid

Ethyl acetate

anhydrous Na₂SO₄.

3 tests are performed for each formulation.

Step 1.

1.1 ml of substrate solution at the concentration of 2.6 mM is placed ina 1.5 ml Corning Costar SpinX tube (Sigma, FIG. 9A), as reported in theFluka commercial assay method. 100 mg of each powdered collagenaseformulation (Formulations 1 and 4), which is hydrated with 4 times itsweight in saline solution, is inserted in the container bearing themembrane with a porosity of 0.22 μm (FIG. 9B). The container ishermetically sealed with a suitable cap to prevent the powder fromswelling due to further absorption of liquid during the experiment. Thecontainer is housed in the tube with the substrate solution (FIG. 9C).The tube is hermetically sealed and inverted to allow contact betweenthe substrate solution and the container membrane. The collagenase andthe substrate can cross the membrane freely, whereas the powders aretrapped in the container. The entire preparation is incubated at 32° C.The containers are replaced as specified in the protocol to simulatedaily application, application every two days and a single application.

For Bionect Start®, which is an ointment, Step 1 is modified asdescribed below:

0.6 ml of substrate solution at the concentration of 1.3 mM is placed in1.5 ml Eppendorf tubes (FIG. 10 : Eppendorf tube for modified step 1),as reported in the Fluka commercial assay method. The caps of theEppendorf tubes are filled with approx. 250 mg of Bionect Start®, whichis placed in contact with the substrate solution and incubated at 32° C.The caps are replaced as specified in the protocol to simulate dailyapplication, application every two days and a single application.

Step 2:

When the pre-set contact period has elapsed, 125 μl of the substratemixture, treated as described in Step 1, is taken up at each fixedtime-point and placed in a 1.5 ml Eppendorf tube. 250 μl of citric acid(25 mM) and 1.25 ml of ethyl acetate are added to this solution. Thesolution is stirred for 15 seconds and centrifuged. The ethyl acetate,which contains the hydrolysed substrate, is transferred to an Eppendorftube containing anhydrous Na₂SO₄. After agitation and centrifugation ofthe Eppendorf tube, the organic phase is transferred to a new Eppendorftube. The hydrolysed substrate contained in the organic phase isdetermined spectrophotometrically at 320 nm.

The data analysis is reported in FIGS. 12-14 .

It is immediately evident that powdered formulations perform far betterthan the reference product at each of the application frequenciestested, and for each time-point considered.

In particular, the values of Formulations 1 and 4 were always highlysignificant compared with the reference product, demonstrating theirbetter efficacy, which means fewer applications for the patient. In anyevent it is obvious that powdered formulations work far better thanthose containing collagenase in a lipophilic carrier. This means notonly that the collagenase present in the powdered formulations acts inthe aqueous environment created when exudate is absorbed by corn starchglycolate, demonstrating the stability in aqueous solution previouslyclaimed, but also that it operates surprisingly better than expected.

As regards the injectable formulations, as previously stated theysubstantially exploit the purity of the enzyme obtained by the processdescribed above and its surprising stability in an aqueous carrier, bothat ambient T and at temperatures ranging between −20 and −80° C. Theinjectable compositions preferably consist of collagenase infreeze-dried form reconstituted in aqueous solution, preferably at thetime of use, and comprise, per unit dose:

-   -   collagenase obtained according to the process described, in a        quantity equivalent to activity of between 120 and 450 nkat;    -   sterile saline solution (NaCl 0.9%);    -   optionally, HA with a weight average molecular weight of between        750 and 1200 kDa, in a concentration ranging between 1 and 30        mg/ml of saline solution, preferably between 8 and 20 mg/ml, and        even more preferably between 10 and 15 mg/ml.

Some examples of preparation of injectable solutions of collagenase willnow be described by way of example but not of limitation.

Example 8: Preparation of an Injectable Solution of Collagenase inSterile Saline Solution (0.9% NaCl)

Collagenase equivalent to 196 nkat Sterile saline solution 1 ml

The enzyme in lyophilic form, contained in a suitable sterile ampoule,is reconstituted with 0.35 ml of sterile saline solution, taken up witha graduated syringe. After gentle stirring a stable solution isobtained.

Example 9: Preparation of an Injectable Solution of Collagenase inSterile Saline Solution (0.9% NaCl)

Collagenase equivalent to 392 nkat Sterile saline solution 1 ml

The enzyme in lyophilic form, contained in a suitable sterile ampoule,is reconstituted with 0.7 ml of sterile saline solution, taken up with agraduated syringe. After gentle stirring a stable solution is obtained.

Example 10: Preparation of an Injectable Solution of CollagenaseFormulated in 750 kDa MW HA

Collagenase equivalent to 196 nkat HA solution 1 ml

The HA solution is preconstituted by dissolving 10 mg of pre-micronisedHA contained in a suitable sterile ampoule in 1 ml of sterile salinesolution. The enzyme in lyophilic form is reconstituted with 0.35 ml ofHA solution, measured with a graduated syringe. After gentle stirringuntil the collagenase powder has dissolved, a stable solution isobtained.

Example 11: Preparation of an Injectable Solution of CollagenaseFormulated in 1200 kDa MW HA

Collagenase equivalent to 392 nkat HA solution 1 ml

The HA solution is preconstituted by dissolving 15 mg of pre-micronisedHA contained in a suitable sterile ampoule in 1 ml of sterile salinesolution. The enzyme in lyophilic form is reconstituted with 0.7 ml ofHA solution, measured with a graduated syringe. After gentle stirringuntil the collagenase powder has dissolved, a stable solution isobtained.

The solutions thus obtained can be stored at 4° C., although it ispreferable to inject them immediately after preparation or within 8hours. In addition to the typically therapeutic uses described above,the collagenase according to the invention can also be used todissociate tissues and isolate cell clusters or single cells, for boththerapeutic and experimental research purposes. This application isused, for example, in the Langerhans islet cell transplantationprocedure to isolate the islet cells from the surrounding pancreatictissue. Collagenase can also be used successfully to isolatecardiomyocytes, hepatocytes and tumour cells for the purpose of thedeveloping the corresponding vaccine, and in general for all cellsusable in the tissue engineering field (bone, cartilage, thyroid, etc).

The invention claimed is:
 1. A method for the treatment of adhesivecapsulitis, Dupuytren's contracture, Peyronie's disease, or cellulitiswhich method comprises: (i) producing and purifying collagenase fromVibrio alginolyticus chemovar. Iophagus by a process comprising thefollowing stages: Stage A: Inoculation of Vibrio Alginolyticus chemovar.Iophagus into an Erlenmeyer flask and fermentation with culture broth ofnon-bovine animal origin; Stage B: Clarification of the fermented broththus obtained by tangential flow ultrafiltration with 100-500 kDMolecular Weight Cut-Off (MWCO) cassettes; Stage C: Dialysis andconcentration of the clarified medium obtained in stage B, by tangentialflow ultrafiltration with 5-30 kD MWCO cassettes; Stage D: Purificationof the solution containing collagenase obtained in Stage C, byanion-exchange resin carrying weak basic groups, at a pH of between 6.9and 7.4; Stage E: Dialysis and concentration of the fractions withcollagenolytic activity 15 collected in Stage D, by tangential flowultrafiltration with 10-50 kD MWCO cassettes; Stage F: Purification ofthe solution thus obtained, by anion-exchange resin carrying strongbasic groups, at a pH of between 6.9 and 7.4; Stage G: Diafiltration andconcentration of the fractions with collagenolytic activity >95%originating from stage F, by tangential flow ultrafiltration with 10-50kD 20 MWCO cassettes; Stage H: Filtration of the solution containingcollagenase thus obtained, through an 0.2 μm absolute filter, andstorage at a temperature of between −20° and −80° C.; and (ii)administering to a patient in need thereof an effective amount of apharmaceutical composition comprising said collagenase; wherein thecollagenase is characterized by: molecular weight 82 kDa; specificactivity between 1000 and 1800 nkat/mg; purity between 98.0 and 100%; nomicrobial or protein contaminants; stability at a pH of between 5.5 and11; stability in aqueous solution at a temperature ranging between 4°and 40° C.; stability in aqueous solution at 4° C. for 30 days stabilityin aqueous solution at a temperature ranging between −20° C. and −80° C.for 24-48 months; lyophilisability to obtain a stable lyophilic powder;and caseinase activity is less than 1.0 U/ml.
 2. The method according toclaim 1, wherein the pharmaceutical composition is injectable.
 3. Themethod according to claim 1, wherein the pharmaceutical compositioncomprises a sterile saline solution.
 4. A method for the treatment ofadhesive capsulitis, Dupuytren's contracture, Peyronie's disease, orcellulitis, which method comprises: (i) producing and purifyingcollagenase from Vibrio alginolyticus chemovar. Iophagus by a processcomprising the following stages: Stage A: Inoculation of VibrioAlginolyticus chemovar. Iophagus into an Erlenmeyer flask andfermentation with culture broth of non-bovine animal origin; Stage B:Clarification of the fermented broth thus obtained by tangential flowultrafiltration with 100-500 kD Molecular Weight Cut-Off (MWCO)cassettes; Stage C: Dialysis and concentration of the clarified mediumobtained in stage B, by tangential flow ultrafiltration with 5-30 kDMWCO cassettes; Stage D: Purification of the solution containingcollagenase obtained in Stage C, by anion-exchange resin carrying weakbasic groups, at a pH of between 6.9 and 7.4; Stage E: Dialysis andconcentration of the fractions with collagenolytic activity collected inStage D, by tangential flow ultrafiltration with 10-50 kD MWCOcassettes; Stage F: Purification of the solution thus obtained, byanion-exchange resin carrying strong basic groups, at a pH of between6.9 and 7.4; Stage G: Diafiltration and concentration of the fractionswith collagenolytic activity >95% originating from stage F, bytangential flow ultrafiltration with 10-50 kD MWCO cassettes; Stage H:Filtration of the solution containing collagenase thus obtained, throughan 0.2 um absolute filter, and storage at a temperature of between −20°and −80° C.; and (ii) administering to a patient in need thereof aneffective amount of a pharmaceutical composition comprising saidcollagenase and hyaluronic acid with a weight average molecular weightranging between 750 and 1200 kDa, in a concentration ranging between 1and 30 mg/ml of saline solution; and wherein the collagenase ischaracterized by: molecular weight 82 kDa; specific activity between1000 and 1800 nkat/mg; purity between 98.0 and 100%; no microbial orprotein contaminants; stability at a pH of between 5.5 and 11; stabilityin aqueous solution at a temperature ranging between 4° and 40° C.;stability in aqueous solution at 4° C. for 30 days; stability in aqueoussolution at a temperature ranging between −20° C. and −80° C. for 24-48months; lyophilisability to obtain a stable lyophilic powder; andcaseinase activity is less than 1.0 U/ml.
 5. The method according toclaim 4, wherein the pharmaceutical composition is injectable.
 6. Amethod for the treatment of adhesive capsulitis, Dupuytren'scontracture, Peyronie's disease, or cellulitis, which method comprisesadministering to a patient in need thereof an effective amount of apharmaceutical composition consisting of 0.35 ml of a sterile salinesolution wherein hyaluronic acid having a weight average molecular 15weight equal to 750 kDa is dissolved at a concentration of 10 mg/ml, andwherein Collagenase enzyme from Vibrio alginolyticus chemovar. Iophagusis reconstituted; wherein the collagenase is characterized by: molecularweight 82 kDa; specific activity between 1000 and 1800 nkat/mg; puritybetween 98.0 and 100%; no microbial or protein contaminants; stabilityat a pH of between 5.5 and 11; stability in aqueous solution at atemperature ranging between 4° and 40° C.; stability in aqueous solutionat 4° C. for 30 days; stability in aqueous solution at a T rangingbetween −20° C. and −80° C. for 24-48 months; lyophilisability to obtaina stable lyophilic powder; and caseinase activity is less than 1.0 U/ml.7. The method according to claim 6, wherein the pharmaceuticalcomposition is injectable.